RheoScale: A tool to aggregate and quantify experimentally determined substitution outcomes for multiple variants at individual protein positions.

Human mutations often cause amino acid changes (variants) that can alter protein function or stability. Some variants fall at protein positions that experimentally exhibit "rheostatic" mutation outcomes (different amino acid substitutions lead to a range of functional outcomes). In ongoing studies of rheostat positions, we encountered the need to aggregate experimental results from multiple variants, to describe the overall roles of individual positions. Here, we present "RheoScale" which generates quantitative scores to discriminate rheostat positions from those with "toggle" (most substitutions abolish function) or "neutral" (most substitutions have wild-type function) outcomes. RheoScale scores facilitate correlations of experimental data (such as binding affinity or stability) with structural and bioinformatic analyses. The RheoScale calculator is encoded into a Microsoft Excel workbook and an R script. Example analyses are shown for three model protein systems, including one assessed via deep mutational scanning. The RheoScale calculator quickly and efficiently provided quantitative descriptions that were in good agreement with prior qualitative observations. As an example application, scores were compared to the example proteins' structures; strong rheostat positions tended to occur in dynamic locations. In the future, RheoScale scores can be easily integrated into computational studies to facilitate improved algorithms for predicting outcomes of human variants.

Encodable activators of SRC family kinases.

There is considerable current interest in the design of encodable molecules that regulate intracellular protein circuitry and/or activity, ideally with a high level of specificity. Src homology 3 (SH3) domains are ubiquitous components of multidomain signaling proteins, including many kinases, and are attractive drug targets because of the important role their interactions play in diseases as diverse as cancer, osteoporosis, and inflammation. Here we describe a set of miniature proteins that recognize distinct SH3 domains from Src family kinases with high affinity. Three of these molecules discriminate effectively between the SH3 domains of Src and Fyn, which are expressed ubiquitously, and two of these three activate Hck kinase with potencies that rival HIV Nef, one of the most potent kinase activators known. These results suggest that miniature proteins represent a viable, encodable strategy for selective activation of Src family kinases in a variety of cell types.